Diagnostic and sample preparation devices and methods

ABSTRACT

Contemplated methods and devices are drawn to preparation and analysis of analytes from biological samples. In a preferred embodiment the analytes are nucleic acids that are both released from biological compartments present in the sample and fragmented through the use of a voltage potential applied to a pair of electrodes. The nucleic acids thus prepared are subsequently characterized.

This application is a division of U.S. patent application Ser. No.14/003,709, filed Dec. 13, 2013, now U.S. Pat. No. 9,580,742, which is aU.S. National Stage Application filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2012/028721, filed on Mar. 12, 2012,which claims priority to and the benefit of U.S. Provisional PatentApplication No. 61/451,528, filed Mar. 10, 2011, each of which is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The field of the invention is compositions and methods related tonucleic acid preparation and analysis, particularly as they relate topoint of care diagnostic devices and methods.

BACKGROUND

Characterization of analytes found in biological samples is an integralpart of both biological research and medical practice. Preparation ofsamples is a common first step in this process, and varies considerablyin complexity depending on the nature of the analyte and the biologicalsample itself. For determination of the concentration of a therapeuticdrug in plasma, sample preparation can be as simple as waiting for aclot to form and transferring a portion of the liquid fraction to ananalyzer. Other analytes, such as nucleic acids, may normally besequestered inside of cells or viruses from which they need to bereleased prior to characterization. In addition, once released it isoften desirable to process such molecules further in order to simplifyanalysis.

Numerous methods are known for releasing analyte molecules from cells,including mechanical approaches (such as sonication, application ofshear forces, application of heat and agitation in the presence ofparticles) and chemical methods (such as the use of heat (to producechemical effect), surfactants, chaotropes, and enzymes). Some of thesehave been adapted for use in cartridge or microfluidics-based platformssuch as those represented by single use in-office and point-of-caretesting devices and, increasingly, as components of complex systemswhere contamination is a concern. For example, U.S. Pat. No. 5,874,046Adiscloses cell disruption through the use of ultrasonics and applicationof shear forces, among other methods. Similarly, GB2416030B disclosesintegrated devices that utilizes mechanical or enzymatic digestion tolyse cells and release their contents for analysis. U.S. Pat. No.6,664,104B2 discloses devices that lyse cells by a variety of meansincluding mechanical disruption, the application of ultrasound, and theaddition of chaotropes to the sample. WO2011/119711A1 discloses an assaycartridge that utilizes elevated temperatures and pressures as a sampletreatment to release analytes from such biological compartments.Unfortunately, mechanical methods for cell disruption can addsignificantly to the complexity of an integrated device and thesupporting instrumentation. Similarly, addition of chemical reagentssuch as chaotropes or enzymes, to a sample not only require a means formetering the correct amount of reagent but may require removal of suchreagents at a later point in the process lest they complicate analysis.This is particularly true for sensitive analytical methods such as PCRor electrochemistry.

Although it is often used for introduction of foreign genetic materialinto cells, electroporation has been shown to be an effective means forthe release of nucleic acids from cells. Release of nucleic acids fromalgal cells has been disclosed (Bahi et al, J. R. Soc. Interface,8:601-608 (2011)), by initially concentrating the cells on an electrodesurface and applying a high frequency (600 kHz) current. Unfortunately,such conditions can lead to the production of considerable heat, whichmay be difficult to dissipate from a microfluidics device without theuse of complex heat exchangers or other thermal transfer devices.US2010/0112667A1 discloses the use of a device with a complex electrodeconfiguration that places numerous small insulators placed between apair of electrodes in order to lyse cells; this configuration generateshigh current field gradients within the small gaps between theinsulators and reduces power requirements. These conditions can alsocause other undesirable effects such as bubbles, increased reactive orinhibiting agents, or breakdown of the supporting structures.

Lysis of eukaryotic cells by generation of hydrolysis products within amicrofluidic device using electrodes has also been described (Di Carloet al, Lab Chip, 5:171-178 (2005), wherein local generation of hydroxideions at relatively high concentrations (estimated at approximately 20mM) were found to be necessary for rapid lysis. These investigators didnot report on recovery or characterization of analytes from the lysedcells. Lee et al (Lab Chip, 10:626-633 (2010)) describe a similar devicethat incorporates an ion exchange polymer diaphragm to generate highhydroxide concentrations in order to effectively lyse bacterial cells.The authors were able to perform PCR on DNA analytes released in thisprocess but noted that this general approach was not suitable for usewith RNA and PCR based detection techniques.

Overall, current methods for release of analytes from cells, viruses,and other biological compartments are difficult to implement in aself-contained microfluidic or point-of-care device. The relativelysmall size of such devices leads to volume restrictions that complicateoperations such as fluid handling related to reagent addition and solidphase capture and release of analytes in order to remove such addedreagents prior to analysis. Their reduced dimension also limit optionsfor applying physical forces (such as ultrasound and shear forces) usingconventional equipment and provide relatively little heat capacity tocontrol temperature. While electrode-based methods such aselectrochemical generation of reactive species have been used, theirutility with certain analytes, notably RNA, is unclear. None of theseapproaches provides controlled analyte fragmentation, which isadvantageous in many direct analytical methods, in addition tobiological compartment lysis.

The above show that there is an unmet need for a method and device thatnot only provides reliable, rapid, easily controlled biologicalcompartment lysis and fragmentation of the analytes thus released, butalso has simple hardware requirements that are readily adaptable tosmall scale devices. Such a device and method may be incorporated into amicrofluidic “lab on a chip” or point-of-care device where controlledcell lysis and analyte fragmentation has utility.

SUMMARY OF THE INVENTION

A modulated electrical potential applied to a set of electrodes was usedto release nucleic acids, and especially RNA, from a biological sample,with the aim of providing a simple and reliable approach to geneticanalysis that was amenable to miniaturization and use in in-office,point-of-care devices. Surprisingly, it was found that control ofvarious aspects of the modulated electrical potential permitted bothrelease of nucleic acids from biological samples and controlledfragmentation of the released nucleic acids (typically RNA). Theobserved fragmentation is achieved rapidly at low voltages, andadvantageously reduces the time required for analysis as well as heatgeneration, reduced bubbles and a reduction in the unwanted chemicalbyproducts. Moreover, such systems and methods are especially desirablewhere downstream analysis is a direct electrochemical RNA hybridizationanalysis.

In one embodiment of the inventive subject matter, a biological sampleor a portion thereof is placed in an extraction zone that includes apair or multiple pairs of electrodes. A modulated potential differenceis applied to the electrodes, which induces release of nucleic acids,and especially RNA, from the biological sample and into solution. Thereleased nucleic acids are fragmented while in the extraction zone, withthe average length of the fragmented nucleic acids being equal to orless than about 75% (more preferably equal to or less than about 50%,and most preferably equal to or less than about 25%) of the length ofthe released nucleic acids prior to fragmentation. The method may havean additional step of adjusting the modulated potential difference, theresidency time of the biological sample in the extraction zone, or bothin order to adjust the average length of the nucleic acid fragments. Themodulated potential difference may be adjusted in a variety of ways,including but not limited to the magnitude of the voltage applied to theelectrodes, the duration of a voltage pulse applied to the electrodes,and the frequency at which a voltage pulse is applied to the electrodes,or a combination of these. In other embodiments, the average length ofthe nucleic acid fragments may be about 200 bases in length or less. Instill other embodiments, the modulated potential difference iscontrolled to produce nucleic acid fragments that have a reduced timerequired for hybridization to a solid-phase probe relative to the timerequired for the nucleic acids to hybridize prior to fragmentation. Insuch embodiments the time required for hybridization of the nucleic acidfragments may be reduced to at least half the time required for thenucleic acids prior to fragmentation. Reagents may be introduced to theextraction zone to improve the efficiency of these processes, suchenhancing reagents can include metal ions, compounds that promote theformation of free radicals, chaotropes, and ionic and nonionicdetergents.

Another embodiment of the inventive subject matter is a device forpreparation of nucleic acids, and especially RNA, from a biologicalsample. Such a device may include a fluid reservoir, a sample receivingzone, and an extraction zone. These may be arranged such that the samplereceiving zone lies between the fluid reservoir and the extraction zoneand is in fluid communication with each, thereby providing a path forfluids from the fluid reservoir, through the sample receiving zone, andinto the extraction zone. In such an embodiment, the fluid reservoir canoptionally have a pliant wall with an external surface, which may beaccessible, and the extraction zone can include a pair or multiple pairsof electrodes. The electrodes of the extraction zone may be configuredto both effect the release of nucleic acids from the biological sampleand to fragment the released nucleic acids. In some embodiments, thefluid reservoir may include an extraction buffer that supports thepreparation of nucleic acids from the biological sample. In someembodiments, pressure applied to the pliant wall of the fluid reservoirdisplaces or deforms the pliant wall into the fluid reservoir, reducingits volume. In such an embodiment, pressure applied to the fluidreservoir causes the fluid stored within to flow into the samplereceiving zone. Such pressure may be applied to the external surface ofthe pliant wall, and may be applied by a device or manually by a user.Biological samples are frequently supplied in the form of a collectingdevice, for example a swab. The sample receiving zone may be configuredto receive and retain such a sample and at least a portion of theattendant collecting device.

In another embodiment of the inventive subject matter, a biologicalsample is analyzed using a device with an extraction zone that includesa pair or multiple pairs of electrodes and is in fluid communicationwith an analysis zone that includes a sensing electrode and a referenceelectrode. A portion of the biological sample is introduced into theextraction zone, where a charge or current is applied to the electrodestherein using a protocol that is effective in releasing nucleic acids,and especially RNA, from the biological sample and fragmenting thereleased nucleic acids to produce fragmented nucleic acids. Thesefragments can have an average length of about 200 bases or less. Thefragmented nucleic acids are moved to the analysis zone, where a secondcharge or current flow has been applied to the sensing electrode and thereference electrode. A third charge or current flow is then measuredbetween the sensing electrode and the reference electrode to quantify,ascertain the presence of, or otherwise characterize a nucleic acid inthe biological sample. In some embodiments, the method includes the useof a sensing electrode that includes a reporting system that isresponsive to nucleic acid hybridization. The sensing electrode mayinclude a probe molecule that is at least partially complementary to afragmented nucleic acid. In some embodiments, the sensing electrode maybe nanostructured, such that the nanostructure is spiky, rough, orfractal. Hybridization time is in part dependent upon the size of thenucleic acid fragments; it is thus advantageous to control the size ofthese fragments. Towards that end, in some embodiments, the first chargeor current flow may be adjusted or modulated to optimize or adjust thelength of the nucleic acid fragments produced in the extraction zone.Such adjustments can include the magnitude of the voltage applied to thepair of electrodes, the duration of a voltage pulse applied to the pairof electrodes, the frequency at which a voltage pulse is applied to thepair of electrodes, the duration of the treatment time or a combinationof these. In other embodiments, the size of the nucleic acid fragmentsmay be optimized or adjusted by controlling residency time of thebiological sample within the extraction zone. In still otherembodiments, both the first charge or current flow to the pair ofelectrodes and the residency time of the biological sample within theextraction zone are adjusted to optimize or adjust the size of thenucleic acid fragments.

Thus, the present inventive subject matter provides for the processingof a biological sample to release and provide nucleic acids, andespecially RNA, in a form suitable for direct analysis. The inventivesubject matter presented herein also provides methods and devices tofragment these nucleic acids in a controlled fashion in order toadvantageously reduce the time required for subsequent analysis. Thesemethods and devices are particularly suitable for incorporation intopoint-of-care devices and microfluidic devices.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts an embodiment of an extraction zone of theinventive subject matter. A pair of electrodes separated by insulatorsare exposed within a reaction space, effecting both cell lysis thatreleases nucleic acids and controlled fragmentation of the nucleic acidsthus released.

FIG. 2 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. A pair of electrodes on a common substratelie within a reaction space defined by a set of insulating walls,effecting both cell lysis that releases nucleic acids and controlledfragmentation of the nucleic acids thus released.

FIG. 3 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. A pair of electrodes lies within aninsulated channel which serves as a reaction space. The electrode paireffect both cell lysis that releases nucleic acids and controlledfragmentation of the nucleic acids thus released.

FIG. 4 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. An array of electrodes lies within aninsulated channel which serves as a reaction space. The array ofelectrodes effects both cell lysis that releases nucleic acids andcontrolled fragmentation of the nucleic acids thus released.

FIG. 5 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. An electrode pair is imbedded in aninsulating matrix. One member of the pair is cylindrical. The remainingelectrode lies within the lumen of the cylindrical electrode, which alsodefines reaction space within which the electrode pair effects both celllysis that releases nucleic acids and controlled fragmentation of thenucleic acids thus released.

FIG. 6 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. Individual electrodes of an electrode pairare attached to insulating substrates that are mounted in parallel, thespace between defining a reaction space within which the electrode paireffects both cell lysis that releases nucleic acids and controlledfragmentation of the nucleic acids thus released. The electrodes includefeatures that project into the reaction space.

FIG. 7 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. Individual electrodes of an electrode pairare attached to insulating substrates that are mounted in parallel, thespace between defining a reaction space within which the electrode paireffects both cell lysis that releases nucleic acids and controlledfragmentation of the nucleic acids thus released.

FIG. 8 schematically depicts another embodiment of an extraction zone ofthe inventive subject matter. A set of porous electrodes form two wallsof a reaction space, which is further defined by a pair of insulatingwalls. Within the reaction space the electrode pair effects both celllysis that releases nucleic acids and controlled fragmentation of thenucleic acids thus released.

FIG. 9 schematically depicts an embodiment of a device of the inventivesubject matter. A reagent reservoir, sample receiving area, andextraction zone are provided on a single substrate. The sample receivingarea is configured to receive the collecting portion of a samplecollector, lies between the reagent reservoir and the extraction zone,and is in fluid communication with both.

FIG. 10 schematically depicts an embodiment of a device of the inventivesubject matter. A reagent reservoir, sample receiving area, extractionzone, and analysis zone are provided on a single substrate. The samplereceiving area is configured to receive the collecting portion of asample collector, lies between the reagent reservoir and the extractionzone, and is in fluid communication with both. A fluid channel directsmaterials from the extraction zone to the analysis zone.

FIG. 11 schematically depicts an embodiment of a device of the inventivesubject matter. A reagent reservoir, sample receiving area, andextraction zone are provided on one substrate. The sample receiving areais configured to receive the collecting portion of a sample collector,lies between the reagent reservoir and the extraction zone, and is influid communication with both. A fluid channel leads from the extractionzone. An analysis zone with a complementary fluid channel is provided ona second substrate.

FIG. 12 schematically depicts an embodiment of the device of theinventive subject matter. A reagent reservoir, sample receiving area,extraction zone, and a plurality of analysis zones are provided on asingle substrate. The sample receiving area is configured to receive thecollecting portion of a sample collector, lies between the reagentreservoir and the extraction zone, and is in fluid communication withboth. A branched fluid channel directs and distributes material from theextraction zone to the analysis zones.

FIG. 13 depicts an embodiment of the device of the inventive subjectmatter. Multiple reagent reservoirs, a sample receiving area configuredto engage a sample collector, an extraction zone, an analysis zone, anda waste collection zone are provided on a single substrate. The deviceis shown engaging a sample swab.

FIG. 14 illustrates results of the disclosed method. FIG. 14A shows astained agarose gel. Lanes 1, 2 and 3 contain size standards; lane 5contains the biological sample prior to treatment; lanes 6 through 10show samples representing different time points during treatment. FIG.14B shows a longer exposure of the gel of FIG. 14A. FIG. 14C provides asummary of experimental conditions.

FIG. 15 is a graph depicting the effect of nucleic acid fragment size onthe time required for analysis.

DETAILED DESCRIPTION

The inventors have discovered that a modulated potential differenceapplied to an electrode pair, or applied independently to a series ofpairs, may be used to both release nucleic acids, and especially RNA,from a biological sample into solution and also fragment the releasednucleic acids in a controlled manner. Release of nucleic acids frombiological compartments such as cells, viruses, spores, and so on intofree solution is necessary prior to characterization by most currentdetection methods, particularly those that rely on hybridization toidentify specific base sequences. Many of these direct analyticalmethods also benefit from fragmentation of the native nucleic acid, andespecially RNA, as this increases the rate of diffusion and speeds thekinetics of sequence specific hybridization. The inventive subjectmatter advantageously supports both of these functions in a singlepreparation area and single processing step, minimizing material lossesand greatly simplifying, and reducing time required for, both workflowand the design of devices that incorporate it. In contrast to chemicaland enzymatic means for releasing and fragmenting nucleic acids, theapplied potential difference is readily controllable. In addition, theinventive subject matter is compatible with known nucleic acidcharacterization methods, for example electrochemical detection, whichmay therefore be readily integrated into the same process or device.While the inventive subject matter may be embodied in a variety ofdevices, its simplicity and minimal hardware requirements make itparticularly suitable for use in compact point-of-care devices ormicrofluidic devices, advantageously permitting the performance ofcomplex genetic characterization on site in a physician's office or inthe field.

As used herein, the term “lysis” refers to the process of disrupting theintegrity of a biological compartment such as a eukaryotic cell, fungus,bacteria, virus, or spore to such an extent that internal components,and especially RNA, are exposed to and may enter the externalenvironment. Examples of lysis include the formation of permanent ortemporary openings in a cell membrane and complete disruption of thecell membrane, both of which permit release of cell contents into thesurrounding solution.

As used herein, the term “analyte” refers to a molecule of interest thata user wishes to characterize, quantify, or verify the presence of.Examples of nucleic acid analytes, and especially ribonucleic acidanalytes, include messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA(miRNA), non-coding RNA, small interfering RNA (siRNA), and transfer RNA(tRNA). Other analytes include DNA, proteins, carbohydrates, and lipids.Still other examples of analytes include low molecular weightmetabolites such as amino acids, nucleotides, and steroids.

As used herein, the term “biological compartment” refers to a discretestructure that contains and segregates biological molecules of interestfrom the surrounding environment, which is typically a liquid media.Examples of biological compartments include, but are not limited to,eukaryotic cells, fungal cells, bacterial cells, spores, pollen,organelles, liposomes, and viruses.

In one embodiment of the inventive subject matter, lysis is performed byapplication of a potential difference to a pair of electrodes that arein electrical contact with a volume of fluid containing a biologicalsample which can contain analytes within a eukaryotic cell, fungus,bacteria, virus, spore, pollen, or other biological compartment.Application of a modulated potential difference to these electrodes,hereafter referred to as lysis electrodes, results in lysis of thebiological compartments and subsequent release of analyte into thesurrounding solution. The potential difference may be modulated in avariety of ways in order to induce lysis of biological compartments froma sample. In some embodiments, a voltage ranging from about 0.5V toabout 3,000V may be applied to the electrodes used for lysis. In apreferred embodiment the voltage applied to the lysis electrodes isabout 40V. This voltage may be constant or may be applied in pulses. Theduration of such voltage pulses can be up to 60 seconds. In a preferredembodiment the duration of a voltage pulse is about 10 milliseconds. Thetime between such voltage pulses can be up to 360 seconds. In apreferred embodiment, the time between voltage pulses is about 1 second.A voltage pulse can also have a characteristic waveform, and may beapplied to the lysis electrodes as a repeating waveform. Voltagewaveforms include, but are not limited to, triangle waves, square waves,sine waves, exponential decaying waves, forward saw tooth waveforms, andreverse saw tooth waveforms. In a preferred embodiment the voltage pulseis applied to the lysis electrodes as a square wave. The mechanism forlysis in this fashion is not known. While not wishing to be bound to anyparticular theory or hypothesis, the inventor contemplated that lysis isa result of electrolytic processes (for example generation of hydroxideand other reactive species) possibly in concert with electroporation.However the use of relatively low frequency, low voltage potentials, andeffectiveness in the presence of buffering species indicates that othermechanisms may be responsible.

In some embodiments of the inventive subject matter, lysis is selective,effecting release of analytes from a subset of cells or other biologicalcompartments present within the biological sample. For example, in suchan embodiment, conditions may be selected to lyse and release analytefrom epithelial cells present in a cheek swab, but not bacterial cellsthat are also present in the sample. Selective lysis may be achieved bycontrolling the voltage that is applied to the lysis electrodes, whichmay range from about 0.5V to about 3,000V. In other embodiments theduration of a voltage pulse that is applied to the lysis electrodes maybe controlled to achieve selective lysis; in such embodiments theduration of the voltage pulse (i.e., the “pulse width”) can range fromabout 1 millisecond to about 60 seconds. In another embodiment selectivelysis can be implemented by controlling the frequency at which voltageis applied to the lysis electrodes; in such embodiments this frequencycan range from about 0.01 Hz to about 1,000 Hz. In yet anotherembodiment, high frequency AC potential, which can range from about 0.1Hz to about 1,000 Hz can be applied to the lysis electrodes in order toeffect selective lysis. In still another embodiment, the duration oflysis treatment may be controlled in order to selectively lyse certaincell or other biological compartment types. In such an embodiment theduration of lysis treatment may range from about 1 millisecond to about5 minutes. In a preferred embodiment two or more of applied voltage,pulse width, voltage frequency, high frequency AC potential, andduration of treatment are controlled in order to perform selectivelysis.

In some embodiments of the inventive subject matter, selective lysis isperformed without the necessity of modulating the potential applied tothe lysis electrodes. In such embodiments, selective lysis may beachieved through the addition of a lysis enhancing probe or sensitizerthat associates with a selected biological compartment type.Alternatively, a lysis depreciating or inhibiting probe or desensitizerthat associates with selected biological compartment types may be usedto effect negative selection. In other embodiments select biologicalcompartments may be segregated prior to lysis, for example byelectrophoretic or dielectrophoretic movement, magnetic capture usingmagnetic particles and capture magnets, antibody-based capturemechanisms, size-selective mechanical filtration, and flow-basedparticle separation mechanisms. In still other embodiments modulation ofthe potential applied to the lysis electrodes may be used in combinationwith the use of one or more of a lysis enhancing probe, a lysisdepreciating or inhibiting probe, or a segregation method in order toprovide selective lysis.

The efficiency of lysis of biological compartments may be improved bythe addition of enhancing reagents to the sample. The enhancing reagentcan be, but not limited to, a metal ion, including iron, ruthenium,zinc, manganese, and/or copper ions. These metal ions may be used incombination with chelating agents (e.g., EDTA). In some embodiments, theenhancing reagent is a compound that supports the formation of freeradicals. Examples of such compounds include, but are not limited to,chelating agents (e.g., EDTA), hydrogen peroxide, organic peroxides, anddissolved oxygen. In another embodiment, the enhancing reagent is achaotropic salt. In yet another embodiment, the enhancing reagent is asurfactant, including nonionic detergents and ionic detergents. In someembodiments, two or more enhancing reagents may be used in combination.

Once lysis is effected, a variety of analytes may be released into thesurrounding media for further analysis. In a preferred embodiment, theanalyte is a nucleic acid. Examples of nucleic acids include DNA, andespecially RNA (e.g., rRNA, mRNA, tRNA, microRNA, noncoding RNA, etc.).Other intracellular analytes, such as proteins (including but notlimited to enzymes, structural proteins, regulatory proteins,cell-surface receptors, and immunoglobulins) may be released in thismanner. Similarly, low molecular weight intracellular analytes (e.g.,nucleotides, hormones, signaling molecules, amino acids, salts, lipids,and steroids) may be released for analysis.

Surprisingly, it was found that, in some embodiments of the inventivesubject matter, analyte molecules that are released from the biologicalcompartments are cleaved or fragmented. For example, RNA from a celllysed by the application of a modulated potential to a pair of lysiselectrodes may have an average length of over 2,000 bases immediatelyupon lysis, but are rapidly cleaved into fragments of reduced lengthunder lytic conditions. The average size of such fragments may be up toabout 75% of the size or length of the unfragmented analyte. In otherembodiments the average size of such fragments may be up to 60%, up to50%, up to 40%, up to 30%, or up to 20% of the size or length of theunfragmented analyte. Thus, in a preferred embodiment, the analyte is anucleic acid (and most typically RNA) where a high proportion of thefragmented nucleic acid is about ≤500 bases, more preferably ≤300 bases,and most preferably ≤200 bases (e.g., between 20 and 100 bases, orbetween 50 and 150 bases) in length. This fragmentation canadvantageously reduce the time required to detect or otherwisecharacterize the released analyte. For example, fragmentation of ananalyte molecule may reduce molecular weight and increase speed ofdiffusion, thereby enhancing molecular collision and reaction rates. Inanother example, fragmenting a nucleic acid may reduce the degree ofsecondary structure, thereby enhancing the rate of hybridization to acomplementary probe molecule. The mechanism for this fragmentation isunclear. It is thought to be a result of electrolytic processes (forexample generation of hydroxide, free radicals, and other reactivespecies), however its effectiveness when relatively low frequency, lowvoltage potentials are used and in the presence of buffering speciesindicates that other mechanisms may be responsible.

Thus, and viewed from another perspective, fragmentation is preferablyadjusted such that subsequent hybridization times are reduced (ascompared to hybridization times for unfragmented nucleic acids underotherwise identical conditions) by at least 25%, more preferably atleast 50%, even more preferably at least 65%, and most preferably atleast 80%. For example, RNA may be released from a cell and fragmentedsuch that the time required for hybridization and electrochemicalanalysis hybridization times is reduced by at least 25%, more preferablyat least 50%, even more preferably at least 65%, and most preferably atleast 80%.

In some embodiments of the inventive subject matter, fragmentation ofthe analyte may be controlled by application of a modulated potentialdifference to the lysis electrodes. The potential difference may bemodulated in a variety of ways. In some embodiments a voltage rangingfrom about 0.5V to about 3,000V may be applied to the lysis electrodes.In a preferred embodiment, the voltage applied to the lysis electrodesis about 100V, or about 200V from peak voltage to peak voltage. Thisvoltage may constant or may be applied in pulses. The duration of suchvoltage pulses can be up to 60 seconds. In a preferred embodiment theduration of a voltage pulse is about 10 milliseconds. The time betweensuch voltage pulses can be up to 360 seconds. In a preferred embodiment,the time between voltage pulses is about 1 second. A voltage pulse canalso have a characteristic waveform, and may be applied to the lysiselectrodes as a repeating waveform. Voltage waveforms include, but arenot limited to, triangle waves, square waves, sine waves, exponentialdecaying waves, forward sawtooth waveforms, and reverse sawtoothwaveforms. In a preferred embodiment, the voltage pulse is applied tothe lysis electrodes as a square wave. In still another embodiment, theduration of lysis/fragmentation treatment may be controlled in order tocontrol the fragmentation of the analyte. In such an embodiment, theduration of lysis/fragmentation treatment may range from about 1millisecond to about 5 minutes. Where the treatment time is the time thefluid is in contact with the electrodes. In a continuous flow device,the total time of lysis for a given sample may be greater than thetreatment times indicated. In some embodiments of the inventive subjectmatter, the potential applied to the lysis electrodes for the lysis ofbiological compartments and for fragmentation of analytes is modulatedin the same manner, such that lysis and fragmentation occur within thesame time frame. In other embodiments, the potential applied to thelysis electrodes is initially modulated to optimize lysis, and thensubsequently modulated to optimize fragmentation of the analyte. In yetanother embodiment, modulated voltages that are optimal forbiocompartment lysis and for analyte fragmentation may be alternated.

In some embodiments of the inventive subject matter, the lysiselectrodes comprise a first electrode and a second electrode separatedby a distance which can range from 1 nanometer to 2 millimeters. Thisspace can contain an insulating material so as to further localize theapplied potential difference to the electrodes. Lysis electrodes may beconstructed of a variety of materials as suits the needs of themanufacturer or application. Suitable materials include carbon andmetals such as gold, silver, platinum, palladium, copper, nickel,aluminum, rhuthenium, and alloys thereof. Suitable materials for lysiselectrodes may also include conductive polymers, including, but notlimited to iodine-doped trans-polyacetylene, poly(dioctyl-bithiophene),polyaniline, metal impregnated polymers and fluoropolymers, carbonimpregnated polymers and fluoropolymers, and admixtures thereof. In someembodiments the lysis electrodes may be made, in whole or in part, of acombination of these materials.

Lysis electrodes may have a variety of geometries and arrangements. Insome embodiments lysis electrodes are mounted on or form part of aninterior surface of a chamber or channel used for lysis (a “lysiszone”). One embodiment is shown in FIG. 1, where one lysis electrode(110) is separated from a second lysis electrode (120) by an insulator(130). The space between the electrodes (140) is occupied, at least inpart, by biological sample during lysis and fragmentation. Anotherembodiment of the inventive subject matter is shown in FIG. 2, wherelysis electrodes (210, 220) lie on an insulating substrate (250) that isexposed to fluid containing a biological sample. In such an embodiment,insulating walls (230) may be used to define a flow channel, and may befurther augmented by the addition of an insulating wall (260) that formsa chamber (240).

In other embodiments, lysis electrodes may lie within the interior spaceof a chamber or channel. For example, FIG. 3 shows a lysis chamber orlysis zone with linear electrodes (310, 320) that lie within a chamberor channel (340) bounded by insulated walls (330). Such a chamber orchannel could be constructed as a serpentine, and configured to utilizeeither a flowing or static sample. FIG. 4 illustrates a similarembodiment that utilizes an array of micro-structured electrodes (410)that are placed in a lysis zone bounded by insulators (420) to form achamber or channel (430) containing a biological sample. In such anembodiment the electrodes may be formed on a planar surface by suitablemeans (including, for example, micromachining, molding, plating, andelectrodeposition) and may be configured such that the gap between theelectrodes is larger than the diameter of an individual electrode.

In other embodiments, the lysis zone is essentially tubular. One suchembodiment is shown in FIG. 5, where one lysis electrode (510) is ahollow tube forming the outer wall of a chamber (540) containing abiological sample and a second lysis electrode (520) lies within thelumen of the tubular electrode (510). The outermost lysis electrode(510) may be surrounded by or embedded in an insulator (530) in such anembodiment. Such an embodiment can be used with either a static orflowing sample.

In some embodiments, the lysis zone is configured as a channelcontaining lysis electrodes. FIG. 6 illustrates such an embodiment,where a channel (640) includes a pair of lysis electrodes (620, 610)that include ridges or projections that protrude into the channel. Theseserve to increase local field strength applied to a biological samplewithin the channel (640) and, in the case of a flowing stream of fluidsample, serve to cause turbulence that improves mixing. Such ridges orprojections may be produced by molding, micromachining,electrodeposition of microstructured materials, or any suitable means.In an alternative embodiment, FIG. 7 shows a channel (740) that includesa pair of lysis electrodes (710, 720) that are configured as a set ofrails that lie the edge of the insulating walls (730) of the channel. Insuch embodiments, the biological sample may be present in a fluid thatflows along the channel or that lies static within the channel.

In yet another embodiment, as shown in FIG. 8, the lysis electrodes(810, 820) are permeable to liquids and are separated by an insulator(830), so that a fluid containing the biological sample within the space(840) that lies between the electrodes may be lysed and fragmented. Thebiological sample may be present in a fluid that flows through the lysiselectrodes or that lies static between the lysis electrodes. In such anembodiment the lysis electrodes may be porous, woven, in the form of amesh or web, or a combination of these, and may also serve as afiltration medium.

The lysis electrode and lysis zone embodiments described above may beincorporated into a cartridge. Such a cartridge may prepare a sample forsubsequent analysis; such a preparative cartridge may, for example, beconfigured to remove a portion of a biological sample from a samplecollector or swab and transport it to a lysis zone where biologicalcompartments are lysed and released analytes fragmented. In such anembodiment, the biological sample may include the contents of an elutedswab in buffer, blood, plasma, serum, cerebral spinal fluid, urine,feces, seminal fluid, mucus, tissue, respiratory fluids, food, water,air, eluted contents of a filter or urogenital secretions. An example ofa preparative cartridge is shown in FIG. 9, where a substrate (910)supports a fluid reservoir (920) that is in fluid communication with asample receiving area or zone (930), which is in turn in fluidcommunication with a lysis chamber or zone (940) that includes a pair oflysis electrodes. A flow of fluid, which can include reagents thatenhance the lysis and fragmentation functions of the device, from thefluid reservoir transports a portion of the biological sample in thesample receiving zone (930) to the lysis zone (940). The fluid reservoir(920) may include a pliant or flexible wall, so that pressure applied tothe outer surface of such a wall reduces the internal volume of thefluid reservoir and induces flow towards the sample receiving zone.Following lysis and fragmentation, the prepared sample can exit thecartridge via an outlet (950).

In other embodiments, a cartridge may be configured to include areassuitable to both prepare a biological sample and characterize theresulting prepared sample, thereby providing a sample-to-answercartridge. An example of a sample-to-answer cartridge is shown in FIG.10. Here, a substrate (1010) supports a fluid reservoir (1020) that isin fluid communication with a sample receiving area or zone (1030),which is in turn in fluid communication with a lysis chamber or zone(1040) that includes a pair of lysis electrodes. A flow of fluid, whichcan include reagents that enhance the lysis and fragmentation functionsof the device, from the fluid reservoir transports a portion of thebiological sample in the sample receiving zone (1030) to the lysis zone(1040). The fluid reservoir (1020) may include a pliant or flexiblewall, so that pressure applied to the outer surface of such a wallreduces the internal volume of the fluid reservoir and induces flowtowards the sample receiving zone. Following lysis and fragmentation,the prepared sample moves to an analysis chamber or zone (1050) that isin fluid communication with the lysis chamber or zone (1040), where itis characterized. Such an analysis zone may include a sensing electrodeand a reference electrode for use in electrochemical detection ofanalytes. In some embodiments, the analysis zone of the analysiscartridge may utilize a lysis electrode of the lysis zone of thepreparation cartridge as a reference electrode. In other embodiments, asingle chamber or flow channel may include both lysis electrodes andsensing and reference electrodes. In still another embodiment, a singlechamber or flow channel may include lysis electrodes and a sensingelectrode, where a lysis electrode serves as a reference electrode foranalyte characterization.

In an alternative embodiment a sample preparation cartridge and ananalysis cartridge may be provided as separate units that are broughtinto fluid communication to form a sample-to-answer device. Thisarrangement advantageously permits alternate configurations and easilycustomizable devices wherein a sample preparation cartridge may becoupled to different types of analytical cartridges to facilely producesample-to-answer devices with different functions or specificities. FIG.11 illustrates such an embodiment, where the sample preparation portionincludes a substrate (1110) that supports a fluid reservoir (1120) thatis in fluid communication with a sample receiving area or zone (1130),which is in turn in fluid communication with a lysis chamber or zone(1140) that includes a pair of lysis electrodes. A flow of fluid, whichcan include reagents that enhance the lysis and fragmentation functionsof the device, from the fluid reservoir transports a portion of thebiological sample in the sample receiving zone (1130) to the lysis zone(1140). The fluid reservoir (1120) may optionally include a pliant orflexible wall, so that pressure applied to the outer surface of such awall reduces the internal volume of the fluid reservoir and induces flowtowards the sample receiving zone. The prepared sample exits the lysiszone via a prepared sample outlet (1150), which may be brought intofluid communication with the prepared sample inlet (1160) of theanalysis cartridge. The analysis cartridge includes a second substrate(1170) that supports a prepared sample inlet (1160) that is in fluidcommunication with an analysis zone (1180) that may include a sensingelectrode and a reference electrode for use in electrochemical detectionof analytes. In some embodiments, the analysis zone of the analysiscartridge may utilize a lysis electrode of the lysis zone of thepreparation cartridge as a reference electrode.

In still another embodiment, a sample-to-answer cartridge is provided inwhich the prepared sample is distributed to two or more analysis zones.This arrangement advantageously permits a variety of characterizationsto be performed on a single biological sample applied to the cartridge.For example, both immunochemical and nucleic acid characterizations maybe performed from the same biological sample on the same cartridge.Alternatively, analysis zones may be configured to perform differentnucleic acid characterization, permitting detection of multiple geneticmarkers from a single biological sample on the same cartridge. Inanother embodiment, one analysis zone may serve as a reference analysiszone. In such an embodiment, the reference analysis zone may beconfigured for characterization of an analyte that has been introducedinto the biological sample or that is known to be present. The result ofsuch a characterization may be used to “scale” the result of acharacterization of a second analyte (in a second analysis zone) that ispresent in unknown quantities, thereby providing a degree of correctionfor performance of the particular device and/or reagents.

Another example of an embodiment with a plurality of analysis zones isshown in FIG. 12. Here, a substrate (1210) supports a fluid reservoir(1220) that is in fluid communication with a sample receiving area orzone (1230), which is in turn in fluid communication with a lysischamber or zone (1240) that includes a pair of lysis electrodes. A flowof fluid, which can include reagents that enhance the lysis andfragmentation functions of the device, from the fluid reservoirtransports a portion of the biological sample in the sample receivingzone (1230) to the lysis zone (1240). The fluid reservoir (1220) mayinclude a pliant or flexible wall, so that pressure applied to the outersurface of such a wall reduces the internal volume of the fluidreservoir and induces flow towards the sample receiving zone. Followinglysis and fragmentation, the prepared sample moves through a branchedfluid channel and is distributed to two analysis zones (1250, 1260),where it is characterized. Each analysis zone may include a sensingelectrode and a reference electrode for use in electrochemical detectionof analytes. Analysis zones may be configured to perform differentcharacterizations. In such an embodiment, multiple analysis zones can beconfigured to utilize a single reference electrode and/or counterelectrode. In an alternative embodiment, analysis zones may utilize alysis electrode of the type previously described in the lysis zone ofthe preparation cartridge as a reference electrode or counter electrode.In other embodiments a single chamber or flow channel may include bothlysis electrodes and sensing and reference electrodes. In still anotherembodiment, a single chamber or flow channel may include lysiselectrodes and sensing electrodes, where a lysis electrode serves as areference electrode for analyte characterization.

In some embodiments of the inventive subject matter, secondary reagentreservoirs are provided that permit the addition of reagents necessaryfor processes occurring downstream from these reservoirs. Reagents maybe supplied as liquids held within the reagent reservoirs but may alsobe supplied as dry reagents that are present in fluid pathways, and arereconstituted when flow of a liquid buffer from an upstream reservoir isestablished. Alternatively, reagents may be supplied as dry reagentsstored with a reagent reservoir. In such an embodiment, dry reagentswould be reconstituted when the user adds liquid, such as buffer orwater, to such a reagent reservoir prior to use.

Fluid pathways of the contemplated devices may include valves to directand control the flow of fluid. For example, in a device in which flow isestablished by applying pressure to a pliant wall that forms part of areagent reservoir one or more one-way valves may be incorporated intothe fluid paths of the device to insure that flexion of the pliant wallon release of pressure does not reverse the direction of flow. In otherembodiments fluid pathways may include bubble trapping features, forexample incorporating a serpentine path in communication with gaspermeable membranes or vents. In some embodiments chambers within thedevice, for example a lysis zone or an analysis zone, may includefeatures that permit verification of an effective level of fluid (e.g.,sufficient to come into contact with the electrodes) within the chamber.For example, a lysis zone and an analysis zone may be constructed, atleast in part, of transparent or translucent materials that permitnoninvasive optical monitoring of the fluid levels within.

As noted above, the analysis zone may be configured to performelectrochemical detection. In such embodiments, the analysis zoneincludes a sensing electrode and a reference electrode. In use, abiasing current or charge is applied to the sensing electrode and thereference electrode. Upon addition of prepared sample, changes in theresulting current or charge is measured to characterize the preparedsample. Such sensing electrodes may be nanostructured, as disclosed inUS2011/0233075, which is incorporated by reference herein. Thenanostructures of the sensing electrode may be rough, spiky, or fractal.Such a sensing electrode may also include a reporting system that isresponsive to a biomolecular stimulus. For example, a reporting systemcould include a probe molecule that is responsive to protein:proteininteractions or to nucleic acid hybridization. Such probe moleculesinclude, but are not limited to, nucleic acids, peptide nucleic acids,morpholino nucleic acid analogs, locked nucleic acids, immunoglobulins,proteins, and peptides. For example, in characterization of a targetnucleic acid from a sample, a probe molecule may include a sequence thatis at least partially complementary to the target nucleic acid'ssequence. In another example, in characterization of an antigen from asample, a probe molecule may include a monoclonal antibody specific forthe antigen. A reporting system may also include tethering portions withchemical groups, such as thiols, that facilitate attachment to thesensing electrode. A reporting system may also include anelectrocatalytic reporter, such as ruthenium hexamine, potassiumferricyanide, or combinations thereof. Such reporting systems mayprovide sufficient sensitivity to directly detect unamplified geneticmaterial from a processed biological sample.

FIG. 13 shows a sample swab and a preferred embodiment of asample-to-answer cartridge. A substrate (1310) supports a fluidreservoir (1320) that is in fluid communication with a sample receivingzone (1340). The biological sample is shown being applied on thecollection tip of a sample collector (1330), which lies within thesample receiving zone (1340), which may be closed using a sample cover(1350). Closing the sample cover both secures the sample applicator anddefines a sample chamber that assists in directing the flow of fluidfrom the fluid reservoir (1320) and through the collection tip of thesample collector (1330), thereby transferring a portion of thebiological sample to the lysis zone (1360). Following lysis andfragmentation at least a portion of the prepared sample is transferredto the analysis zone (1380); secondary reagents from a secondary reagentreservoir (1370) may be added at this time. Waste materials arecollected in a waste reservoir (1390) as the biological sample isprepared and characterized.

There are a number of formats, materials, and size scales that may beused in the construction of the sample preparation and sample analysiscartridges described herein. Some embodiments are constructed, at leastin part, as microfluidic devices. In such embodiments, the reagentreservoirs, lysis zones, analysis zones, and the connecting fluidchannels may be comprised of PDMS (or similar polymers), and fabricatedusing soft lithography.

In some embodiments, single layer devices are contemplated. In otherembodiments multilayer devices are contemplated.

Other methods of fabrication are of single and multilayer devices are,but not limited to, micromachining of bulk solid, use of pressuresensitive adhesives with channel structures cut and subsequentlylaminated, injection molded, overmolded, thermo formed or hot embossedstructures, or any other method that is used in manufacture ofmicrofluidic or larger structures known to those skilled in the art.Examples

Lysis and Fragmentation. A suspension of Escherichia coli was preparedin nuclease-free PBS and introduced into a processing zone containing apair of lysis electrodes. A 40V potential was applied to the lysiselectrodes as 40 millisecond pulses at a frequency of 1 Hz. Samples wereprepared at various time intervals and applied to a 2% agarose gelprepared with 1×TBE, along with appropriate size standards, then stainedwith SYBR Gold and imaged. Results are shown in FIG. 14. FIG. 14A showsa normal exposure of the gel; FIG. 14B shows the same gel that has beenoverexposed in order to reveal detail. Lanes 1 and 2 contain high andlow molecular weight standards (respectively), lane 3 contains arepresentative 20 mcr oligo, and lane 4 contains a total E. coli RNAcontrol. A sample taken prior to the application of voltage to the lysiselectrodes was placed in lane 5, which does not show significantmaterial entering the gel. Lanes 6 through 10 show the effect of voltageapplied to the lysis electrodes for 25, 50, 75, 100 and 125 seconds fromthe initiation of voltage. These devices were run with 40 ms pulse widthand a frequency of 1 Hz. Lanes 6-10 equate to a cumulative appliedvoltage of 1, 2, 3, 4 and 5 s respectively. Significant release of highmolecular weight RNA is apparent with as little as 1 second of appliedvoltage to the lysis electrodes. Release of additional RNA andfragmentation of the RNA so released is apparent after as little as twoseconds, with the steady accumulation of RNA fragments of less thanabout 500 bases in length and an accompanying loss of high molecularweight RNA as the total applied voltage increases to 5 seconds. FIG. 14Cprovides a key for the experimental lanes of the gel.

Effects of RNA Fragmentation. A simulation was performed to determinethe effect of reduction in RNA analyte size due to fragmentation on thetime required for sample analysis. The time required to accumulate amolecule of RNA on a sensing electrode was calculated as a function ofthe size of the target RNA present in a processed sample. Results areshown in FIG. 15. Reduction in the size of the analyte RNA from 2000bases to 500 bases or less in length reduces the time required toaccumulate, and therefore analyze, the analyte RNA by 50% or more.

Thus, specific embodiments and applications of methods and devices forsample preparation and analysis have been disclosed. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Furthermore, where a definition or use of a termin a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

What is claimed is:
 1. A device for analyzing a biological sample, thedevice comprising: a sample receiving zone configured to receive abiological sample, the biological sample including nucleic acids; asample extraction zone in fluid communication with the sample receivingzone and configured to receive the biological sample from the samplereceiving zone, the sample extraction zone including a plurality ofelectrodes in electrical communication with an electrical source andconfigured to fragment the nucleic acids into a plurality of fragmentednucleic acids when a first electrical signal is applied to the pluralityof electrodes; a signal control unit configured to determine an overalltreatment time of the first electrical signal applied to the pluralityof electrodes based on a desired average length in bases of theplurality of fragmented nucleic acids; and a sample analysis zone influid communication with the sample extraction zone and including asensing electrode and a reference electrode for analyzing the biologicalsample.
 2. The device of claim 1, further comprising: a fluid reservoirincluding a pliant wall defining an interior volume, the pliant wallhaving an external surface configured such that pressure applied to theexternal surface of the pliant wall results in deformation of the pliantwall into an interior volume of the fluid reservoir, thereby inducingfluid flow through the sample receiving zone.
 3. The device of claim 2,wherein the fluid reservoir includes an extraction buffer.
 4. The deviceof claim 1, further comprising: a flow channel, wherein each of theplurality of electrodes extends along a length of the flow channel, andwherein each of the plurality of electrodes comprises ridges orprojections that protrude into the flow channel.
 5. The device of claim1, wherein each of the plurality of electrodes is disposed in theextraction zone and configured to effect release of a plurality ofnucleic acids from the biological sample and to effect fragmentation ofthe plurality of nucleic acids.
 6. The device of claim 1, wherein thesample receiving zone is configured to receive and retain the biologicalsample and at least a portion of a sample collecting device.
 7. Thedevice of claim 1, wherein the sensing electrode includes a probemolecule that is at least partially complementary to the fragmentednucleic acid.
 8. The device of claim 7, wherein the sample analysis zoneincludes a reporter system that is responsive to nucleic acidhybridization to the probe molecule.
 9. The device of claim 8, whereinthe sensing electrode includes a nanostructured microelectrode, whereinthe nanostructured microelectrode is at least one of textured andfractal.